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RESEARCH ARTICLE SPECIAL COLLECTION: TRANSLATIONAL IMPACT OF RAT The role of the dopamine D1 receptor in social cognition: studies using a novel genetic rat model Judith R. Homberg 1, *, Jocelien D. A. Olivier 2, * , , Marie VandenBroeke 3 , Jiun Youn 3 , Arabella K. Ellenbroek 3 , Peter Karel 1 , Ling Shan 1 , Ruben van Boxtel 4 , Sharon Ooms 1 , Monique Balemans 1 , Jacqueline Langedijk 1 , Mareike Muller 1 , Gert Vriend 5 , Alexander R. Cools 1, , Edwin Cuppen 4 and Bart A. Ellenbroek 3 ABSTRACT Social cognition is an endophenotype that is impaired in schizophrenia and several other (comorbid) psychiatric disorders. One of the modulators of social cognition is dopamine, but its role is not clear. The effects of dopamine are mediated through dopamine receptors, including the dopamine D1 receptor (Drd1). Because current Drd1 receptor agonists are not Drd1 selective, pharmacological tools are not sufficient to delineate the role of the Drd1. Here, we describe a novel rat model with a genetic mutation in Drd1 in which we measured basic behavioural phenotypes and social cognition. The I116S mutation was predicted to render the receptor less stable. In line with this computational prediction, this Drd1 mutation led to a decreased transmembrane insertion of Drd1, whereas Drd1 expression, as measured by Drd1 mRNA levels, remained unaffected. Owing to decreased transmembrane Drd1 insertion, the mutant rats displayed normal basic motoric and neurological parameters, as well as locomotor activity and anxiety-like behaviour. However, measures of social cognition like social interaction, scent marking, pup ultrasonic vocalizations and sociability, were strongly reduced in the mutant rats. This profile of the Drd1 mutant rat offers the field of neuroscience a novel genetic rat model to study a series of psychiatric disorders including schizophrenia, autism, depression, bipolar disorder and drug addiction. KEY WORDS: Dopamine D1 receptor, Mutant rat, Social cognition, Characterization, Schizophrenia INTRODUCTION It is now commonly agreed that psychiatric disorders are not fully independent syndromes, but rather represent constellations of symptom clusters that partially overlap. In support of this idea, there is extensive comorbidity between psychiatric disorders. For instance, patients with schizophrenia often show symptoms of major depressive disorder, autism spectrum disorders, bipolar disorders and/or drug addiction. Intriguingly, a very recent report analysing structural magnetic resonance imaging (MRI) studies has reported that schizophrenia, and several major psychiatric disorders are jointly characterized by changes in brain areas mediating social cognition (Goodkind et al., 2015). The social brainis indeed a common factor in schizophrenia and its comorbid disorders (Fett et al., 2015; Hoertnagl and Hofer, 2014; Pinkham, 2014). Social cognition can be defined as all processes that are elicited by and/or directed towards other subjects (Kennedy and Adolphs, 2012). Increasing effort has been made to understand the intricacies of the social brain (Kennedy and Adolphs, 2012; Meyer-Lindenberg and Tost, 2012). One of the major players is dopamine. Research in non-human primates and rodents has shown that amphetamine, a dopamine-releasing drug, reduces affiliative social behaviour (Ellenbroek et al., 1989; Schiorring, 1979). However, it has also been reported that pro-social behaviour (induced by playing back 50 kHz ultrasonic vocalizations that elicit approach behaviour) is associated with increased dopamine release (Willuhn et al., 2014). These conflicting data might in part be related to opposing roles of different dopamine receptors. In this study, we focus on the role of the dopamine D1 receptor (Drd1). A recent positron emission tomography (PET) scan study in healthy volunteers showed that Drd1 binding was positively correlated to social conformity (Plaven- Sigray et al., 2014). In line with this, studies in zebrafish have found that Drd1 antagonists reduced social preference (Scerbina et al., 2012). By contrast, studies in male macaque monkeys have shown that a Drd1 antagonist actually improved social behaviour that was disrupted by amphetamine (Ellenbroek et al., 1989). Likewise, local injections of a Drd1 antagonist into the nucleus accumbens of mice leads to increases in social approach behaviour in females but not males (Campi et al., 2014). Finally, Drd1 agonists injected into the nucleus accumbens of male prairie voles prevent new pair bonding, but facilitated the maintenance of pair bonding when the agonist was administered after a bond was formed (Aragona et al., 2006). Taken together, these data emphasize that, although Drd1 clearly plays an important role in social cognition, its precise function is far from understood. The conflicting data might at least in part be due to differences in the behavioural paradigm being used, as well as differences in species and sex. Perhaps most importantly, most studies investigating the role of Drd1 in social cognition have relied on pharmacological manipulations of Drd1. Unfortunately, none of the drugs acting on Drd1 show selectivity for Drd1 over other dopamine receptors, and several of these drugs also show affinity for serotonin receptors (Undieh, 2010). Therefore, in order to investigate the role of Drd1 in social cognition as a transdiagnostic marker for schizophrenia and comorbid psychiatric disorders, we introduce in the present paper Received 1 February 2016; Accepted 4 May 2016 1 Donders Institute for Brain, Cognition and Behaviour, Department of Cognitive Neuroscience, Radboud University Medical Centre, Nijmegen 6525 EZ, The Netherlands. 2 Department of Neurobiology, Unit Behavioural Neuroscience, Groningen Institute for Evolutionary Life Sciences, University of Groningen, Groningen 9700 CC, The Netherlands. 3 Victoria University of Wellington, School of Psychology, PO Box 600, Wellington 6040, New Zealand. 4 Hubrecht Institute, KNAW and University Medical Centre Utrecht, Utrecht 3584 CT, The Netherlands. 5 CMBI, Radboud University Nijmegen Medical Centre, Geert Grooteplein 2628, Nijmegen 6525 GA, The Netherlands. *These authors contributed equally to this work Deceased Author for correspondence ( [email protected]) J.R.H., 0000-0001-9108-7188; J.D.A.O., 0000-0003-4654-9763 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1147 © 2016. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2016) 9, 1147-1158 doi:10.1242/dmm.024752 Disease Models & Mechanisms

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  • RESEARCH ARTICLE SPECIAL COLLECTION: TRANSLATIONAL IMPACT OF RAT

    The role of the dopamine D1 receptor in social cognition: studiesusing a novel genetic rat modelJudith R. Homberg1,*, Jocelien D. A. Olivier2,*,‡, Marie VandenBroeke3, Jiun Youn3, Arabella K. Ellenbroek3,Peter Karel1, Ling Shan1, Ruben van Boxtel4, Sharon Ooms1, Monique Balemans1, Jacqueline Langedijk1,Mareike Muller1, Gert Vriend5, Alexander R. Cools1,†, Edwin Cuppen4 and Bart A. Ellenbroek3

    ABSTRACTSocial cognition is an endophenotype that is impaired in schizophreniaand several other (comorbid) psychiatric disorders. One of themodulators of social cognition is dopamine, but its role is not clear.The effects of dopamine are mediated through dopamine receptors,including the dopamine D1 receptor (Drd1). Because current Drd1receptor agonists are not Drd1 selective, pharmacological tools are notsufficient to delineate the role of theDrd1. Here, we describe a novel ratmodel with a genetic mutation in Drd1 in which we measured basicbehavioural phenotypes and social cognition. The I116Smutation waspredicted to render the receptor less stable. In line with thiscomputational prediction, this Drd1 mutation led to a decreasedtransmembrane insertion of Drd1, whereas Drd1 expression, asmeasured by Drd1 mRNA levels, remained unaffected. Owing todecreased transmembrane Drd1 insertion, the mutant rats displayednormal basic motoric and neurological parameters, as well aslocomotor activity and anxiety-like behaviour. However, measures ofsocial cognition like social interaction, scent marking, pup ultrasonicvocalizations and sociability, were strongly reduced in the mutant rats.This profile of the Drd1 mutant rat offers the field of neuroscience anovel genetic rat model to study a series of psychiatric disordersincluding schizophrenia, autism, depression, bipolar disorder and drugaddiction.

    KEY WORDS: Dopamine D1 receptor, Mutant rat, Social cognition,Characterization, Schizophrenia

    INTRODUCTIONIt is now commonly agreed that psychiatric disorders are not fullyindependent syndromes, but rather represent constellations ofsymptom clusters that partially overlap. In support of this idea,there is extensive comorbidity between psychiatric disorders. For

    instance, patients with schizophrenia often show symptoms ofmajor depressive disorder, autism spectrum disorders, bipolardisorders and/or drug addiction. Intriguingly, a very recent reportanalysing structural magnetic resonance imaging (MRI) studies hasreported that schizophrenia, and several major psychiatric disordersare jointly characterized by changes in brain areas mediating socialcognition (Goodkind et al., 2015). The ‘social brain’ is indeed acommon factor in schizophrenia and its comorbid disorders (Fettet al., 2015; Hoertnagl and Hofer, 2014; Pinkham, 2014). Socialcognition can be defined as all processes that are elicited by and/ordirected towards other subjects (Kennedy and Adolphs, 2012).Increasing effort has been made to understand the intricacies of thesocial brain (Kennedy and Adolphs, 2012; Meyer-Lindenberg andTost, 2012). One of the major players is dopamine.

    Research in non-human primates and rodents has shown thatamphetamine, a dopamine-releasing drug, reduces affiliative socialbehaviour (Ellenbroek et al., 1989; Schiorring, 1979). However, it hasalso been reported that pro-social behaviour (induced by playing back50 kHz ultrasonic vocalizations that elicit approach behaviour) isassociated with increased dopamine release (Willuhn et al., 2014).These conflicting data might in part be related to opposing roles ofdifferent dopamine receptors. In this study, we focus on the role of thedopamine D1 receptor (Drd1). A recent positron emissiontomography (PET) scan study in healthy volunteers showed thatDrd1 binding was positively correlated to social conformity (Plaven-Sigray et al., 2014). In line with this, studies in zebrafish have foundthat Drd1 antagonists reduced social preference (Scerbina et al.,2012). By contrast, studies in male macaque monkeys have shownthat a Drd1 antagonist actually improved social behaviour that wasdisrupted by amphetamine (Ellenbroek et al., 1989). Likewise, localinjections of a Drd1 antagonist into the nucleus accumbens of miceleads to increases in social approach behaviour in females but notmales (Campi et al., 2014). Finally, Drd1 agonists injected into thenucleus accumbens of male prairie voles prevent new pair bonding,but facilitated the maintenance of pair bonding when the agonist wasadministered after a bond was formed (Aragona et al., 2006).

    Taken together, these data emphasize that, although Drd1clearly plays an important role in social cognition, its precisefunction is far from understood. The conflicting data might – atleast in part – be due to differences in the behavioural paradigmbeing used, as well as differences in species and sex. Perhaps mostimportantly, most studies investigating the role of Drd1 in socialcognition have relied on pharmacological manipulations of Drd1.Unfortunately, none of the drugs acting on Drd1 show selectivityfor Drd1 over other dopamine receptors, and several of thesedrugs also show affinity for serotonin receptors (Undieh, 2010).Therefore, in order to investigate the role of Drd1 in socialcognition as a transdiagnostic marker for schizophrenia andcomorbid psychiatric disorders, we introduce in the present paperReceived 1 February 2016; Accepted 4 May 2016

    1Donders Institute for Brain, Cognition and Behaviour, Department of CognitiveNeuroscience, Radboud University Medical Centre, Nijmegen 6525 EZ,The Netherlands. 2Department of Neurobiology, Unit Behavioural Neuroscience,Groningen Institute for Evolutionary Life Sciences, University of Groningen,Groningen 9700 CC, The Netherlands. 3Victoria University of Wellington, School ofPsychology, PO Box 600, Wellington 6040, New Zealand. 4Hubrecht Institute,KNAW and University Medical Centre Utrecht, Utrecht 3584 CT, The Netherlands.5CMBI, Radboud University Nijmegen Medical Centre, Geert Grooteplein 26–28,Nijmegen 6525 GA, The Netherlands.*These authors contributed equally to this work†Deceased

    ‡Author for correspondence ( [email protected])

    J.R.H., 0000-0001-9108-7188; J.D.A.O., 0000-0003-4654-9763

    This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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    © 2016. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2016) 9, 1147-1158 doi:10.1242/dmm.024752

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    http://dmm.biologists.org/collection/rat-disease-modelmailto:[email protected]://orcid.org/0000-0001-9108-7188http://orcid.org/0000-0003-4654-9763http://creativecommons.org/licenses/by/3.0http://creativecommons.org/licenses/by/3.0

  • a novel rat model with a mutation in the Drd1 gene. We firstprovide evidence that the mutant Drd1 is dysfunctional.Subsequently, we measured basic behaviour and social cognitionin the rat carrying the Drd1 mutation. To assess social cognitionwe used social interaction, social approach and avoidance,olfactory-scent marking and social anxiety (maternal-separation-induced ultrasonic vocalizations) tests. Our data show that thereceptor Drd1 is crucially involved in all these aspects of socialcognition. As these social traits are also prominent inschizophrenia and comorbid disorders, we believe our rat modelto be useful for research into these disease states.

    RESULTSWe treated male Wistar rats with N-ethyl-N-nitrosourea (ENU) toinduce mutagenesis (Smits et al., 2006) and identified a rat amongthe offspring with a missense mutation in the gene encoding forDrd1. The mutation involves a hydrophobic isoleucine into polarserine residue exchange (Drd1I116S) in helix III at position 116 ofthe protein. We outcrossed this mutant rat for at least fivegenerations to wild-type Wistar rats to generate a Drd1 mutant ratmodel. We characterized and tested this mutant rat model for basicbehavioural phenotypes and social cognition.

    Characterization of the Drd1I116S mutant ratComputational modelling of the Drd1I116S receptorWe first performed a computational analysis and modelling of theDrd1I116S mutation. The ENU-induced mutation changed ahydrophobic isoleucine residue into a hydrophilic serine residueat position 116. The Drd1 protein belongs to the D1-like family of

    dopamine receptors that couples to the intracellular G proteinG-stimulatory (Gs). As illustrated in Fig. 1, computationalmodelling predicted that, in Drd1I116S, the cytoplasmic ends ofhelix III and helix VI cannot approach each other as closely as in thewild-type Drd1, breaking contact with the highly conserved R120 atthe extracellular side of helix III that binds to G proteins. Theseparation of the cytoplasmic ends of helix III and helix VI mightopen this part of the receptor to a larger extent, meaning that thereceptor could be rendered instable.

    Dopamine receptor mRNA levelsNext, to assess whether the Drd1I116S mutation affects Drd1transcript levels, we determined mRNA expression of Drd1 in thestriatum (rich in dopamine) by quantitative PCR (qPCR). We alsoassessed mRNA levels of other dopamine receptor subtypes (Drd2–Drd5), to evaluate whether a change in Drd1 would indirectly affectthe expression of genes encoding for other dopamine receptors. Wefound that the mRNA levels of Drd1 (Z=−1.389, P=0.165), Drd2(Z=−0.579, P=0.563), Drd3 (Z=−0.000, P=1.000), Drd4 (Z=−0.347, P=0.728) and Drd5 (Z=0.000, P=1.000) were unaltered inthe striatum of wild-type rats compared to Drd1I116S mutant rats(Fig. S1). Drd4 mRNA levels were very low in both wild-type andmutant rats, which corresponds to previous findings for Drd4mRNA levels in the striatum of wild-type subjects (Matsumotoet al., 1996). None of the dopamine receptors showed any differencein levels between the wild-type and Drd1 mutant genotypes,suggesting that the Drd1I116S mutation does not affect Drd1transcript stability and/or degradation, nor that of the otherdopamine receptors.

    Fig. 1. Computational modelling of thewild-type Drd1and the Drd1I116S protein structure. A computationalmodel of (A) the wild-type Drd1 receptor, and the (B)Drd1I116S receptor is shown. The solid arrow indicates theposition of the I116S mutation. The purple amino acidsreflect helix VI, and the red amino acids reflect helix III.Ligand binding takes places at about two to three helicalturns above the solid arrow. The I116S mutation (locatedjust inside the membrane near the cytosolic side of helixIII) breaks a series of hydrophobic contacts with aminoacids 276 and 277 (valine and isoleucine) in helix VI andwith the highly conserved R120 at the extracellular side ofhelix III. The exact position of this arginine residue, andthe contacts between helices III and VI are important formaintaining the receptor in an inactive state. The I116Smutant disturbs the position of R120 and the interactionsbetween helices III and VI. This might lead to a repulsionof helix VI (broken arrow), an opening for G-proteinbinding (lower side of the displayed protein) andactivation and instability of the receptor.

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    RESEARCH ARTICLE Disease Models & Mechanisms (2016) 9, 1147-1158 doi:10.1242/dmm.024752

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    http://dmm.biologists.org/lookup/doi/10.1242/dmm.024752.supplemental

  • Drd1I116S receptor bindingTo measure whether the Drd1I116S mutation would affect Drd1ligand binding we used [3H]SCH23390 (an Drd1 antagonist)autoradiography, focusing on the whole brain. This methodmeasures both transmembrane and intracellular Drd1 binding,given that cells are cut at varying positions when slicing the brain.We found that [3H]SCH23390 binding was reduced by ∼20%(olfactory tubercle) to ∼50% (prefrontal cortex, subcortical regions,substantia nigra) in the Drd1I116S mutant compared to wild-type rats(Fig. 2A; Table S1). Thus, Drd1 binding is significantly reduced inDrd1I116S mutant rats. Because Drd1 mRNA levels were unaltered,and protein synthesis presumably is unaltered as well, it is mostlikely that reduced Drd1 binding reflects reduced Drd1transmembrane insertion.

    Transmembrane insertion of the Drd1I116S receptorBecause receptor instability might affect transmembrane insertionof Drd1I116S, we measured Drd1I116S cell surface expression usingan in vitro system. We cloned the wild-type and Drd1 mutantreceptor with a hemagglutinin epitope (HA) tag into COS7 cells. Byusing an anti-HA antibody we could identify the cellular position ofthe Drd1 under cell impermeable (transmembrane Drd1identification) and permeable (intracellular Drd1 identification)conditions. We found that mutant Drd1 receptor expression wasreduced in ‘live’ and impermeable COS7 cells compared to wild-type Drd1 receptor expression (Fig. 2B). The ‘fixed’ andpermeabilized cells revealed that Drd1 expression in thecytoplasm was equal for the mutant and wild-type Drd1. No Drd1immunoreactivity was observed in cells that were transfected withan empty vector. The data imply that the Drd1 mutation affects Drd1stability and thereby its transmembrane insertion.

    Drd1I116S receptor functionTo assess Drd1I116S function in vivo, we measured the effects of theD1 antagonist SCH23390 on motor reflexes in the paw test(Ellenbroek et al., 1987), a behavioural test known to be sensitive toD1 receptor antagonists. Given that the data were not normallydistributed, the Mann–Whitney U-test was used to assess statisticaldifferences between treatments. In wild-type rats the Drd1antagonist SCH23390 significantly increased the forelimbreaction time (FRT; Z=−2.742, P

  • Because bodyweight was significantly reduced in Drd1I116S ratswe measured food intake in their home cage. We found that regulareating measured over four subsequent days in the home cage(Fig. S3) was significantly decreased in Drd1I116S rats [t(1,6)=5.947,P

  • results for the second and third phase are shown in Fig. 6. Given thatthe data were normally distributed, a mixed model ANOVA withgenotype as between and zone as within subject design was used.The data show that in phase 2, although therewas a significant effectof zone [in proximity of the cup; F(1,32)=9.7, P0.9) for the time spentin each arm (Fig. 6A). However, with respect to zone entries, there

    was a significant zone [Fig. 6B; F(1,32)=10.8, P

  • showing a much more restricted pattern (Fig. 7A), as compared to amore diffuse pattern in theDrd1I116Smutant rats (Fig. 7B) around thesocial stimulus. There were no clear differences in the scent markingpatterns around the non-social stimulus (data not shown).Statistically, this led to a significant genotype×stimulus interactionfor clustering [F(1,1022)=4.2; P

  • Drd1I116S mutant rats was less functional compared to wild-typerats. Drd1 mutant rats did not display gross anatomical changes,although their bodyweight was significantly reduced. Nodifferences were observed in general motor, sensory andneurological functions. Likewise exploratory behaviour andanxiety-like behaviour were not affected by the Drd1I116S

    mutation, except for rearing. The adapted SHIRPA test revealedan increase in vocalization upon handling of the rats, hintingtowards a role of the Drd1 in the social domain. In this line, severalaspects of social cognition were significantly reduced in theDrd1I116S mutant rats.Computational modelling and analysis of the Drd1I116S mutation

    suggested crucial alterations in the mutant rat model. Owing to theDrd1I116S mutation, the cytoplasmic ends of helix III and helix IVcannot approach each other as closely as in the wild-type receptor.Therefore, this part of the receptor might be opened up to a largerextent, making it more accessible to G proteins. The Drd1I116S

    mutation is also located closely to the DRY motif, which isimportant for interaction with intracellular G proteins, andmutations in this motif often lead to constitutive activity (Fanelliet al., 2009). Unfortunately, we have been unable to collect evidence(e.g. Drd1 agonist-induced cAMP production) for constitutiveactivity of the receptor. A potential reason is that, due to themutation, the receptor quickly disassembles upon Drd1 agonistbinding. These changes are also predicted to lead to lowermaximum attainable activity due to decreased transmembraneinsertion of the receptor (Ringkananont et al., 2006). To confirmthis speculation Drd1 binding was measured in an ex vivo system by[3H]SCH23390 autoradiography in the whole brain. We found thatDrd1 binding was reduced by 20–50% in Drd1I116S mutant rats.However, autoradiography does not discriminate betweentransmembrane and intracellular Drd1 binding. To resolve this, weconducted an in vitro experiment in which we overexpressed wild-type and mutant Drd1 in COS7 cells, allowing us to assess thetransmembrane insertion of the wild-type and mutant Drd1. Wefound that particularly transmembrane Drd1 insertion was reduced,with limited changes in intracellular Drd1 expression. This suggeststhat the Drd1I116S mutation affects Drd1 stability and itstransmembrane insertion, whereas the intracellular Drd1expression remains unaltered. Reduced transmembrane insertionof Drd1 likely has consequences for Drd1 function. Indeed, in thepaw test, we found that Drd1I116S mutant rats were less responsive to

    the selective Drd1 receptor antagonist SCH23390. Although it is notyet fully clear how the Drd1I116S mutation affects Drd1, the datasuggest that the Drd1I116S mutant rat model represents a model forreduced Drd1 function.

    We used the adapted SHIRPA test for basic phenotyping of the ratmodel. This test did not reveal motor, sensory and neurologicalconsequences of the Drd1I116S mutation. We did observe asignificantly reduced bodyweight in Drd1I116S mutant rats. Giventhat Drd1 agonists stimulate the secretion of growth hormone fromthe pituitary (Bluet-Pajot et al., 1990), reduced bodyweight mightbe explained by reduced Drd1 function. Another possibility is thatreduced food intake might cause reduced bodyweight. Of note, weobserved that food intake in the home cagewas reduced in Drd1I116S

    mutants, but this difference disappeared when food intake wascorrected for bodyweight. Most likely, the reduced foodconsumption reflects the lower calorific requirements of a smallerbody. Given that animals were weight-matched in the socialinteraction experiments, it is not likely that decreased bodyweightinfluenced the differences in social behaviour.

    Because dopamine plays a crucial role in exploratory behaviour,we assessed this type of behaviour in the rats when exposed to acircular and square open field, as well as the elevated plus maze.Although exploratory behaviour was not affected in these novelenvironments, Drd1I116S mutant rats showed decreased rearing inthe circular open field. Reduced rearing has been linked to decreasedlocomotor activity (Görisch and Schwarting, 2006; Thiel et al.,1999). However, we did not observe a decrease in locomotoractivity in Drd1 mutant rats. In previous work, a decrease in rearingwas also related to a decrease in food foraging, which mightcorrespond to the reduced food consumption in Drd1I116S mutantrats. The absence of exploratory changes in the elevated plus mazeindicates that there were no genotype differences in anxiety-likebehaviour. This was supported by the lack of genotype differencesin self-grooming, which in novel environments can be seen as astress-related displacement behaviour. Given that the previouslyreported relationship between Drd1 and self-grooming (Wachtelet al., 1992) is almost exclusively based on the effects of Drd1agonists and antagonists, and because – without exception – allDrd1 ligands also bind to the Drd5, our results imply that Drd5 ismore involved in self-grooming than Drd1. Taken together, thesedata indicate that general exploratory activity and anxiety levels arenormal in Drd1I116S mutant rats and do not confound behaviouralmeasures in Drd1I116S mutants.

    Social cognition covers a wide variety of components, many ofwhich are disturbed in schizophrenia and other psychiatricdisorders. Social withdrawal, as measured in the social interactiontest, is considered as a measure of the negative symptoms ofschizophrenia (Wilson and Koenig, 2014). Withdrawal from socialcontact might derive from a lack of desire to have social contact. Associal interactions are able to induce conditioned place preference(El Rawas et al., 2012), the social withdrawal might be explained byanhedonia, the inability to experience pleasure or reward. Thisexplanation is supported by the scent marking test, in which malerats are allowed to approach and scent female urine, and mark thescents by urination. Given that sniffing female urine is highlyrewarding for male rats (Malkesman et al., 2010), it is possible thatscent marking around female urine marks was strongly reduced inDrd1I116S rats due to an inability to experience female urine reward.Although we did not specifically investigate olfaction in theDrd1I116S rats, the fact that there was no significant differencebetween the total number of scent markings between the genotypessuggests that olfaction per se was not affected. The reduction in

    Fig. 8. Comparison of ultrasonic vocalizations made by wild-type andDrd1I116Smutant rats. The number of ultrasonic vocalizations made by 7-day-old wild-type (WT) and Drd1I116S mutant (MUT) rats during a 5-min separationfrom the mother (n=8 for each genotype). Drd1I116S mutant rats calledsignificantly less frequently compared to wild-type rats, which might reflect areward deficit. Data display (mean+s.e.m.) of ultrasonic vocalizations.***P

  • scent markings around the social scent (and especially the alteredpattern and reduced saturation) suggest that the effects arespecifically related to a deficit in social cognition.Most mammalian infants, including rat pups, vocalize when

    isolated. Given that interactions with adult females just beforeisolation increase vocalizations (a process termed maternalpotentiation), it is thought that the vocalization reflects a markerof pup–mother social bonds (Shair, 2014). Expression of thematernal potentiation of the ultrasonic vocalization in pups ishypothesized to be related to reward processes, in part becausedopamine activity plays a regulatory role. It has been demonstratedthat activation of dopamine type-2 receptors in the nucleusaccumbens blocks maternal potentiation without alteringvocalization rate in an initial isolation (Shair, 2014). By contrast,it has been found that Drd1 agonists, but not antagonists, reduce thenumber of isolation-induced infant rat ultrasonic vocalizations(Dastur et al., 1999; Muller et al., 2009), with the latter study evenreporting a significant increase with the D1 antagonist SCH23390.Here, we demonstrate that Drd1 dysfunction reduced the totalnumber and duration of isolation-induced rat pup ultrasonicvocalizations. At present it is difficult to explain these differences,though it should be remembered that D1 agonists and antagonistsare non-selective and also affect other receptors. Moreover, whereasin the present study ultrasonic vocalizations were measured atpostnatal day 7, Muller and colleagues used 11- or 12-day-old pups.As ultrasonic vocalizations depend strongly on the age of theanimals (Scattoni et al., 2009), this might (partly) underlie thedifferences between the studies. Regardless of this, the currentfindings are in line with the results from the other social paradigmsand might reflect a reward deficit, hindering the formation of a bondwith the mother. Potentially, this could lead to less maternal care(not measured in the present study) and feeding, and aberrantdevelopment of social behaviour.Finally, sociability and social novelty as measured in the social

    approach and avoidance test were affected by the Drd1I116S

    mutation. Whereas no genotype differences in duration of socialapproach and avoidance were found, the Drd1I116S mutant ratsshowed a significant reduction in the frequency of visiting the areaaround the novel pup in phase 2. Likewise, in phase 3, whereas thewild-type rats visited the novel rat significantly more frequently thanthe familiar rat, this behaviour was not seen in the Drd1I116S mutantrats. Analysis of the social approach and avoidance test usuallyincludes both total time spent in the vicinity of the cylinders andtotal time sniffing the cylinder, with the latter being the moresensitive measure (Silverman et al., 2010). In this respect, it isimportant to realize that in our analysis (using Ethovision XT) welimited the analysis to the nose point, which is closely correlated tosniffing. Thus, Drd1I116S mutant rats likely showed deficits in socialsniffing. This is reminiscent of the finding of the social interactiontest, where we found a significant reduction in active, but notpassive, social behaviour in the Drd1I116S mutant rats.This study provides an initial characterization of a novel Drd1

    mutant rat model, with clear endophenotypes in the social domain.Besides that the present findings raise new research questions to beaddressed in future studies, we also like to mention some otherpotential limitations of our study. Most importantly, due to ENUmutagenesis, the Drd1I116S mutant rats might bear additionalmutations that have not been characterized. However, given thatanimals were outcrossed for at least five generations, the chance ofadditional mutations occuring is reduced to

  • exposed to one test only. All experiments were conducted with the approvalof the Animal Care Committee of the Radboud University in Nijmegen andthe Victoria University of Wellington, according to the respective laws forexperimental animals. All efforts were made to minimize the amount ofanimals and their suffering. Rats were not randomized, because groups weredetermined by genotype. Ex vivo and in vitro experiments were conducted ina blinded fashion by coding the materials. In vivo experiments could not beblinded, because wild-type and Drd1I116S mutant rats can easily bediscerned visually due to the lower bodyweight of the latter.

    GenotypingGenomic DNA was isolated from ear cuts that were sampled in a 96-deepwell block (2.5 ml Riplate, Ritter) and dissolved overnight at 55°C in 300 µllysis buffer (100 mM Tris-HCl pH 8.5, 200 mM NaCl, 0.2% SDS, 5 mMEDTA and 100 µg/ml freshly added proteinase K). Tissue debris was spundown for 15 min at 15,000 g and supernatant was transferred to fresh tubes.DNA was precipitated by adding an equal volume of isopropanol, mixingand centrifugation at 15,000 g at room temperature. The supernatant wasremoved by gently inverting the block and the pellets werewashed with 70%ethanol, and dissolved in 400 µl water. Genotyping was performed using theKASPar SNP Genotyping System (KBiosciences, Hoddesdon, UK) andgene-specific primers (two allele-specific oligonucleotides of ∼40 nt inlength and one common oligonucleotide of ∼20 nt in length). Briefly, aPCR was carried out using the optimal thermocycling conditions for KTaq(94°C for 15 min; 20 cycles of 94°C for 10 s, 57°C for 20 s and 72°C for40 s; GeneAmp9700, Applied Biosystems, Foster City, CA). The PCRcontained 2 μl DNA solution, 1 μl 4× reaction mix, 15 pM reverse primerand 15 pM forward primer, 0.025 μl KTaq polymerase solution and 22 mMMgCl2 in a total volume of 4 μl. Samples were analysed in a PHERAstarplate reader (BMG Labtech, Offenburg, Germany) and data were analysedusing Klustercaller software (KBiosciences). All genotypes were confirmedin an independent reaction.

    Computational analysis and modellingHomology models of the inactive and active form of the Drd1 were obtainedfrom the G-protein-coupled receptor data base (GPCRDB; Vroling et al.,2011). These models were built using the YASARA software (Krieger et al.,2002) using the protocol as described in Krieger et al. (2009) and alignmentsas provided in the GPCRDB. The structure with protein data bank (PDB)identifier 1gzm was used as template for the Drd1 in the inactive state,whereas the structure with PDB identifier 3sn6 was used as template for theactive form.

    qPCRTissue punching, RNA preparation and cDNA synthesisSnap-frozen brain samples from rats (eight wild-type and eight Drd1I116S

    mutant rats) were partly defrosted and the striatum was bilaterally punchedusing a 1.2-mm punching needle (Paxinos and Watson, 2004). Brain tissuesamples were homogenized with 1000 μl QIAzol Lysis Reagent (QIAGENSciences, MD) and 200 μl chloroform (Merck, Darmstadt, Germany). RNAwas isolated with an RNeasy Lipid Tissue Mini Kit (Qiagen, 74804)according to the manufacturer’s protocol. RNA concentration and qualitywas determined with a Nanodrop TMND-1000 spectrophotometer (ThermoFisher Scientific). The samples were kept at −80°C until the next day, whencDNA was made. cDNA was made with an iScript cDNA synthesis Kit(Bio-Rad, Hercules, CA) according to the manufacturer’s protocol.

    qPCR procedureThe quantitative PCR (qPCR) procedures have been described previously(Shan et al., 2012). In brief, qPCR was performed in a reaction volume of20 µl, using the SYBR Green PCR kit (Promega, Madison, WI) and amixture of sense and antisense primers (2 pmol/µl). Primers used for qPCRare shown in Table S3. Reactions were run in aGeneAmp 7300 thermocyclerunder the following conditions: 2 min at 50°C and 10 min at 95°C, followedby 40 cycles of 15 s at 95°C and finally 1 min at 60°C. Data were acquiredand processed automatically by the Applied Biosystems Sequence DetectionSoftware. Specificity of amplification was checked by means of meltingcurve analysis and electrophoresis of products on a 1.5% agarose gel. Sterile

    water (non-template control) and omission of reverse transcriptase (non-RTcontrol) during cDNA synthesis served as negative controls.

    Amplification efficiency was determined by running qPCRs on a dilutionseries of pooled cDNA from all the subjects. Resulting cycle threshold (Ct)values were plotted against the inverse log of each dilution and the slope ofthis curve was then used to calculate the efficiency as follows: efficiency(E)=10−(1/slope). The normalization factor was based upon the geometricmean of the following four reference genes selected by geNorm analysis(Vandesompele et al., 2002): tubulin-α (TUBA), tubulin-β4 (TUBB4),glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ubiquitin C(UBC). To minimize the variation, all qPCRs were conducted in duplicate.

    Quantitative autoradiographyRats were killed by decapitation, their brains rapidly removed, frozen inliquid nitrogen and stored at −80°C. Coronal sections (16 µm) were cut on acryostat microtome at −20°C, thaw-mounted onto gelatin-coated slides andstored at−20°C until use. Frozen sections were brought to room temperatureat 60 min prior to the assay. The tissue sections (at least four slices peranimal and two animals per genotype) were pre-incubated for 20 min atroom temperature in 50 mMTris-HCL buffer at pH 7.4, containing 120 mMNaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM EDTA, 10% w/vBSA, and 1 mM ascorbate. Subsequently, the sections were incubated infresh buffer containing 1 nM [3H]SCH23390 (85.0 Ci/mmol, GEHealthcare, UK) and 40 nM ketanserin (blocking 5-HT2 receptors), in thepresence or absence of unlabelled 2 µM butaclamol (nonspecific binding)for 1 h at room temperature. The slides were then rinsed in cold 50 mMTris-HCl buffer for 15 s to remove superfluous radioligand, washed (4×5 min),rapidly dipped in cold distilled water and dried under a cold stream of air.[3H]SCH23390 sections together with [3H]Microscales™ standards (GEHealthcare, UK) were opposed to a [3H]hyperfilm (GE Healthcare, UK) andmanually developed after 1 month. The [3H]hyperfilms were scanned usinga 9200 typhoon scanner (GE Healthcare, UK). The areas of interest weredetermined using the Paxinos and Watson rat brain atlas, 6th edition (http://labs.gaidi.ca/rat-brain-atlas/). For each brain area, a fixed size square orrectangle box was placed in the area of interest across all slices. Using thetyphoon scanner, the average pixel density within the boxes was measured.This was also done for the [3H]Microscales™ standards that were used in thestandard curve. The optical densities within the brain regions of interest wereconverted into fmol/mg of tissue equivalent using this standard curve. Non-specific binding was subtracted from total [3H]SCH23390 binding.

    In vitro Drd1 mutant overexpression studiesWild-type and mutant Drd1 was N-terminally fused to a hemagglutininepitope (HA) tag, cloned into an expression vector pcDNA3.1 (Invitrogen)and expressed in COS7 cells. At 24 h after transfection, cells were eitherfixed with methanol or incubated in cold DHB medium (DMEM, 25 nMHEPES and 0.2% fatty acid-free BSA) on ice for 20 min followed by 1-hincubation on ice with rabbit 1:200 anti-HA antibody (ab9110, Abcam,Cambridge, UK) in DHB medium and methanol fixation. Samples wereincubated for 1 h at room temperature with blocking buffer (1%BSA in PBSwith 0.1% Tween 20). Cells were immediately fixed after the transfectionprocedure and then incubated for 1 h at room temperature with 1:200 rabbitanti-HA antibody (Abcam) in blocking buffer. All cells were washedwith PBS three times, incubated with 1:200 goat FITC-conjugated anti-rabbit-IgG antibody in blocking buffer for 1 h in the dark. After threewashes with PBS, coverslips were mounted using Vectashield with DAPI(Brunschwigchemie, Amsterdam, The Netherlands) and cells were analysedusing confocal microscopy. The cells were checked for mycoplasmacontamination, and found to be negative.

    Basic behavioural characterization of the Drd1I116S mutant ratsAdapted SHIRPAThe SHIRPA protocol was developed as a quick screen for general measuresof health, motoric and neurological parameters in genetically modifiedanimals. The procedure described by Rogers et al. (1997) was modified forthe rat. Rats were placed in a Perspex jar on a grid for 5 min and evaluated forbody position, spontaneous activity, respiration, body tremor and number offecal boli. Immediately thereafter, rats were placed in a wooden box with

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  • walls (57×57×28 cm). The floor contained separated back line drawings of16 squares. The following behaviours were recorded: transfer arousal,latency time to walk to the walls, locomotor activity, eye opening, gait,particularities of the body hair, pelvic elevation, tail position, approachreaction and touch escape. The rats were then lifted by the tail and assessedfor the position at which struggling movements occurred, as well as trunkcurl, head righting reflex and clenching with the paws to the tail. At landingon the horizontal grid, the rats were evaluated for grasping the grid bar, gripstrength when dragged by the tail across the grid, body tone after fingercompression on each side, pinna, corneal andwhisker reflexes, aswell as toe-pinch withdrawal of the hind limb after squeezing with hand-held forceps.The forepaws were then placed on a horizontal bar and the ability of the ratsto hang suspended was evaluated. Furthermore parameters like skin colour,hind limb tone, abdominal tone, lacrimation, salivation and provoked bitingin response to a pair of tweezers being put in their mouth were observed. Theair-righting reflex, together with contact righting when placed inside a smallplastic tubewere evaluated followed by the postural effects. Finally, rats wereplaced on a horizontal grid, rotated toward a vertical position of 45° andassessed for negative geotaxis, and behaviours like freezing behaviour,irritability, aggression and vocalization were scored. For all rats, thebodyweight, nose-to-tail length and rectal body temperature was measured.

    Food consumption in the home cageRats were housed in genotype-matched pairs and the wirebar lids containingstandard food pellets were weighed during four consecutive days. In order tocorrect for bodyweight rats were weighed on the first day of measuring asbodyweight was not expected to change during this short period.

    Paw testThe paw test was developed to assess dopaminergic functioning andconsists of a Perspex platform (30×30×20 cm) containing four holes, twohindlimbs holes (diameter: 5 cm), two forelimbs holes (diameter of 4 cm)and a slit for the tail (Ellenbroek et al., 1987). The distance between the leftand right forelimb and hindlimb holes was 15 mm, and the distance betweenforelimb and hindlimb holes was 55 cm. At 30 min before testing the ratswere treated with SCH23390 (1 mg/kg bodyweight, intraperitonealinjection). The rats were placed on the platform by inserting thehindlimbs and subsequently the forelimbs in the holes. Hind limbretraction time (HRT) and forelimb retraction time (FRT) were measured,with a minimum of 1 s and a maximum of 30 s. The test was repeated at 40and 50 min after injection. The three measurements were averaged.

    Novelty-induced exploratory behaviour and self-groomingRats were placed in a transparent circular open field (diameter 20 cm) for30 min, and behaviour was videotaped. Exploratory behaviour, rearing, theduration and frequency of total grooming, and face and body grooming wereanalysed using Observer (Version 3.1, Noldus Information Technology,Wageningen, The Netherlands). Additionally, another group of animals wastested in a square open field measuring 50×50×50 cm for 30 min.Locomotor activity (distance moved, cm) was monitored usingEthoVision (Version 3.1, Noldus Information Technology).

    Elevated plus-maze testThe apparatus was made of grey PVC and elevated 75 cm above the floor.The four arms (50×10 cm2) formed a cross with the central platform. Awall(height: 30 cm) of non-transparent material enclosed two arms, locatedopposite to each other. Each rat was placed on the central platform facing oneof the enclosed arms and allowed to freely explore the maze for 5 min.Behaviour was scored manually using Observer 5.0 (Noldus InformationTechnology, Wageningen, The Netherlands). The time spent on the openarm of the maze was calculated as a measure of anxiety, whereas the totalnumber of closed arm entries was considered as ameasure of general activity.

    Assessment of social behaviour in the Drd1I116S mutant ratsSocial interactionAdult rats were isolated for 3 days prior to the experiment to enhance thedisplay of social behaviour. On the day of the experiment, pairs of rats (ofsimilar age, sex and genotype) from different litters were placed in a

    standard translucent polypropylene box (40 cm×72 cm×22.5 cm high),under dim light conditions. The beddings of dust-free wood chips from bothsubjects’ cages were combined and transferred to the experiment box inorder to reduce the novelty of the environment. The behaviour of therats was recorded for 30 min with a video camera mounted above thecage. Duration and frequency of the following behaviours was scoredoffline using Observer® (Noldus Information Technology, Wageningen,The Netherlands) (blinded for genotype): (1) active social behaviour, bothrats are in close proximity with each other and at least one of the rats isactively investigating (sniffing, grooming etc.) the other rat; (2) passivesocial behaviour, both rats are in close proximity (within ∼5 cm of eachother) but neither animal shows active social investigation; (3) non-socialbehaviour, rats are further than 5 cm away from each other.

    Social approach and avoidanceSocial approach avoidance was measured in a T-maze (arms 50 cm long,20 cm wide, with 25-cm-high walls) using a three-phase paradigm. In thefirst phase (habituation), rats were placed in the T-maze and allowed toexplore the maze. After 15 min the animal was removed and the mazecleaned with 70% ethanol. Next, two small cylindrical cups (9 cmdiameter) were placed upside down at the end of each of two arms. Underone cup a juvenile rat was placed, while the other cup was empty. Theexperimental rat was placed back and was able to explore the cups for5 min. After this second phase, the rat was removed and a new juvenile ratwas placed under the previous empty cup, while the familiar pup stayed inthe previous cup. The experimental rat was placed back again and for afinal 5 min was allowed to explore the T-maze. The behaviour of theexperimental rats was measured using Ethovision XT v.9. This programallows for a detailed tracking of behaviour, detecting nose-point, centralbody-point and tail-base point separately. For this analysis, the total timethe nose-point was within a zone of 5 cm of each of the cylinders wasanalysed. In addition, the frequency of (nose-point) zone visits wasanalysed.

    Scent markingScent marking was measured using a protocol very similar to that developedfor mice (Wohr et al., 2011). Briefly, adult male rats were familiarized withfemale rats (from the same genotype) by placing them together with afemale for a period of 5 min between 5 and 7 days before the experiment, toallow them to experience female scent. On the day of the experiment, ratswere allowed to habituate to a novel round open field (diameter 80 cm).After 15 min the experimental rat was removed and the open field cleaned.Two circular pieces of filter paper (diameter 30 cm) were placed in oppositequadrants of the open field, one impregnated with 30 μl of lemon scent(non-social stimulus) and one with 30 μl of fresh urine (social stimulus)from females in oestrus. Females that were in oestrus (as determined by avaginal smear; Marcondes et al., 2002) were gently held between theforelimbs. This was usually sufficient to induce urine flow. This urine wascollected in Eppendorf tubes and used within 1 h after collection.

    After both filter papers were impregnated with the smells, theexperimental male rat was placed back and allowed to explore the openfield for an additional 5 min. After this period the animal was removed, thefilter papers sprayed with ninhydrin spray (which dyes amino acids) anddried overnight. The male urine scent markings left on the filter paper sheetswere analysed by an open source software (openCFU) designed to countsmall circular objects (for the details of the program, see Geissmann, 2013).To differentiate between normal micturition and scent markings (which aremuch smaller), a maximum filter size of 10 pixels was applied to the imageprocessing. The variables analysed were the number of male urine markingssurrounding the target scent, the size of each marking (radius), saturation(intensity of each marking) and clustering (the number of markings in closeproximity to another marking).

    Ultrasonic vocalizations in young ratsUltrasonic vocalizations were recorded in 7-day-old male pups. Rat pupsfrom different litters were taken from their mother and placed in a smallcircular container (diameter 9 cm) with fresh bedding material on the floor.Ultrasonic vocalizations were recorded for a period of 5 min from individual

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  • pups using Ultravox XT®. The same program was used to count all callswithin the 30–50 kHz range. Both the total number of calls and totalduration were recorded.

    Statistical analysesData were checked for normality and homogeneity and analysed usingStudent’s t-tests (home-cage food consumption, novelty-inducedexploratory behaviour, self-grooming and elevated plus maze), mixedmultivariate tests (social interaction and ultrasonic vocalizations), mixedmodel ANOVA with repeated measures (social approach and avoidance,scent marking), a χ-squared test (adapted SHIRPA) or a Mann–WhitneyU-test (qPCR and paw test). All data were analysed using SPSS 16.0software (LEAD technologies, Chicago, IL). No a priori power analysis wasconducted; because of the novelty of the rat model, a priori data for a poweranalysis were not available. The level of significancewas set at P

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